Hybrid Fluorescent Mass-Tag Nanotrackers as Universal Reagents for Long-Term Live-Cell Barcoding

Barcoding and pooling cells for processing as a composite sample are critical to minimize technical variability in multiplex technologies. Fluorescent cell barcoding has been established as a standard method for multiplexing in flow cytometry analysis. In parallel, mass-tag barcoding is routinely used to label cells for mass cytometry. Barcode reagents currently used label intracellular proteins in fixed and permeabilized cells and, therefore, are not suitable for studies with live cells in long-term culture prior to analysis. In this study, we report the development of fluorescent palladium-based hybrid-tag nanotrackers to barcode live cells for flow and mass cytometry dual-modal readout. We describe the preparation, physicochemical characterization, efficiency of cell internalization, and durability of these nanotrackers in live cells cultured over time. In addition, we demonstrate their compatibility with standardized cytometry reagents and protocols. Finally, we validated these nanotrackers for drug response assays during a long-term coculture experiment with two barcoded cell lines. This method represents a new and widely applicable advance for fluorescent and mass-tag barcoding that is independent of protein expression levels and can be used to label cells before long-term drug studies.


Equipment
All mass cytometry experiments were performed using a Helios updated CyTOF2 (DVS Sciences, Fluidigm Co., CA, USA) except for titrations of metal-labeled antibodies which were performed in a CyTOF2 (DVS Sciences, Fluidigm Co., CA, USA). Flow cytometry experiments were performed on a FACSCanto II system (Becton Dickinson & Co., NJ, USA). The analysis of the results obtained in both cytometers was done using the Flowjo® 10 software and Cytobank. Cell viability was assayed using an M200 Nanoquant microplate reader to measure absorbance. Confocal microscopy images were obtained using a Zeiss LSM 710 confocal laser scanning microscope and Zeiss ZEN 2010 software for image acquisition. Transmission electron microscopy experiments were carried out using an ultra-high resolution FEI Titan G2 microscope with a XFEG Field Emission Gun operating at 300 kV. XPS spectra were obtained using a Kratos Axis Ultra-DLD X-ray photoelectron spectrometer equipped with an Al monochromatic X-ray source, over powdered nanoparticle samples.
The mixture was stirred under argon for 1 h before heating to 68 °C for 15 h. Nanoparticles (NPs) were obtained by centrifugation (11,000 x g, 15 min) and washed with methanol (2 x 10 mL) and water (2 x 10 mL). Finally, NPs were stored in water (10 mL) at 4 °C 1 .

Characterization of NK-NPs (1) 2.4.1. Solid content (SC) of the emulsion (%)
A known mass of a suspension of polystyrene NPs (0.5-1mg, suspended in water) was placed in a watch glass, covered with aluminum foil, dried at 25 °C for 15 h, weighed and reweighed to give the mass of NPs. The solid content was then calculated according to the following equation: Equation S1 . Solid content of NK-NPs (1).
CS: 3%, 3 mg of NPs in 100 μL of solution. Where N: Number of particles/g for dry powder, ρ: Density of solid spheres (g/cm 3 ), which is 1 g/cm 3 for polystyrene, d: Mean diameter (nm).

Calculation of number of particles per gram
Result: N= 2.77 x 10 13 NTs per gram

Calculation of loading of NPs using Fmoc NPs test
Fmoc-(x)-NPs (where x is Fmoc-PEG-OH or Fmoc-Lys(Dde)-OH, etc) were resuspended in 1 mL of 20% piperidine in DMF (3 x 20 min) after which the beads were washed three times by centrifugation, and the supernatants combined. Loading was calculated according to the following equation: . Amino-loading of nanoparticles.

Qualitative ninhydrin test
Reaction completion control was determined by qualitative ninhydrin test (Kaiser test) 2 . Small samples of NPs (12 µL, 3% SC) were transferred to 0.5 mL capacity eppendorf and washed once with methanol and centrifuged after which 6 µL of reagent A and 2 µL of reagent B were added. The suspension was mixed well and heated to 100 ºC for 3 min. Blue stained resin beads indicate the presence of primary amines.

Determination of NPs concentration (NPs/µL) by spectrophotometric method
NPs concentration (NPs per microliter) was determined by a spectrophotometric method as described previously 3 . Briefly, measurement of turbidity optical density at 600 nm of polystyrene NP suspensions was performed, based on nephelometric principles. Light going through NP suspensions is scattered via reflection, refraction and diffraction phenomena and the intensity of the scattered light, which is proportional to number of NPs in suspension, is recorded by standard spectrophotometers. In this way, calibrate standard curves were obtained for amino-methyl cross-linked polystyrene NPs of 410 nm by NP known concentrations. Calibration curves fitted linear regression models by which the number of NPs per microliter corresponding to one unit of OD600 for each size could be determined. Thus, these curves using initial batches of NP suspensions permitted us to estimate the number of NPs in final batches, which underwent multiple handling procedures, by OD600 measurement of 1 µL (Figure S10).  Figure S10. Calibration standard curve of concentration of nanoparticles (OD 600).

Synthesis of nanotrackers 3.1. Synthesis of Cy5/Cy3-NTs (3)
NK-NPs (1) nanoparticles were first conditioned by washing them three times with 1 mL of DMF each time through suspension-centrifugation cycles (13,400 rpm, 3 min). Next, to conjugate the PEG spacer to NTs, Fmoc-PEG-OH (15 eq) was dissolved in DMF (1 mL) with oxyma (15 eq) and DIC (15 eq). The mixture was stirred at RT for 10 min. Then, the solution was added to dry NTs, and the suspension was left to stir at 1,400 rpm at 60 °C for 2 h. Subsequently, NTs were washed with three successive suspension-centrifugation cycles (13,400 rpm, 3 min) to obtain Fmoc-PEGylated NTs. Fmoc was removed using a 20% piperidine/DMF solution (3 × 20 min) and sequential washing steps, as described above. Next, 50 µL of a sulfo-Cy5/Cy3-NHS ester solution (1 eq) was mixed separately with DIPEA (1 eq) before being added to dry PEGylated NTs, which were then suspended. The suspension was stirred at 1,000 rpm at RT for 14 h in the dark. Afterwards, the NTs were washed with three cycles of suspension-centrifugation with DMF (13,400 rpm, 3 min), then other three cycles in MeOH, and finally resuspended in mQ H2O to obtain Cy5-NTs (3A) and Cy3-NTs (3B).